METHODS FOR TREATMENT AND PREVENTION OF OTOTOXICITY BY siRNA

ABSTRACT

The present invention relates to methods for reducing and/or preventing ototoxicity caused by an ototoxic agent, noise or head and/or neck radiation. It is also directed to a method for preventing or reducing generation of reactive oxygen species in the inner ear of a patient. The methods of the present invention include administering at least one siRNA directed against TRPV1 mRNA, NOX3 mRNA or a combination thereof.

FIELD OF THE INVENTION

The present invention is directed to a method for preventing and/orreducing ototoxicity in a patient caused by an ototoxic agent, noise orhead and neck radiation, by silencing a TRPV1 gene in the ear of apatient, e.g., by using a siRNA directed against TRPV1, administering asiRNA directed against NOX3, or a combination thereof. The presentinvention also relates to a method for preventing or reducing generationof reactive oxygen species in the inner ear of a patient byadministering to the patient at least one siRNA directed against TRPV1mRNA and/or NOX3 mRNA.

BACKGROUND OF THE INVENTION

There are three major causes of hearing loss: noise-dependent hearingloss, drug-associated hearing loss and age-associated hearing loss.Interestingly, there appears to be a common mechanism to all three majorcauses of hearing loss, namely destruction of sensory epithelium andcochlear neurons through reactive oxygen species. In terms of treatment,no efficient drug treatment or prophylaxis of hearing loss are availableat this point and the only option at present is the use of hearing aids.Tinnitus, also referred to as phantom hearing, is a common and in someinstances invalidating medical complaint. Presently, the pathophysiologyof the disease is poorly understood and there is no proven causativetreatment available. There is however evidence that reactive oxygenspecies might play a role in the pathophysiology of tinnitus.

With respect to the drug-associated hearing loss, prolonged exposure ofthe cochlear cells to aminoglycosides is linked to the killing of outerhair cells in the organ of Corti and type I sensory hair cells in thevestibular organ, leading to permanent hair loss and vestibular damage.Damage to the hair cells progresses from the base of the cochlea (anarea for high frequency sound detection) to the apex (an area for lowfrequency sound detection). This is followed by retrograde damage to theauditory nerve. The degree of hair cell damage and hearing loss isdirectly proportional to the dose of the drug to which the hair cellsare exposed. Repeated exposure to aminoglycosides leads to an additivedamage to hair cells and other structures and subsequently to deafness.Damage is more significant in the elderly who may have less hair cell orlower endogenous protective mechanisms or in other individuals withcompromised auditory function. In addition, damage is generallypotentiated by the concurrent administration of diuretics, such asethacrynic acid and furosemide, which produce reversible hearing loss bythemselves.

Furthermore, platinum containing drugs also cause ototoxicity andthereby hearing loss. Several reports have concluded that the generationof reactive oxygen species (ROS) is linked to cisplatin ototoxicity(reviewed by Forge and Schacht, 2000). Hearing loss due to cisplatin isusually permanent and cumulative. TRPV1 is a member of the transientreceptor potential (TRP) channel family, expressed primarily by smalldiameter neurons (Aδ and C fibers) comprising the pain pathway. It is anonselective cation channel which demonstrates responsivity to heat(Caterina et al., Nature 389, pp. 816-824, 1997). TRPV1 receptorexpression has also been demonstrated in non-neuronal tissues includingorgan of Corti, keratinocytes and bladder urothelium (Zheng J et al. J.Neurophysiol. 90, 444-455, 2003; Southall et al., J. Pharmacol. Exp.Ther., 90, 444-455, 2003), suggesting additional roles in addition tothe regulation of thermal pain sensation. The organ of Corti representsa major site for cisplatin induced hearing loss, a common side effect ofthis antineoplastic agent (Rybak, 1999). The mechanism underlying thehearing loss appears to involve the generation of reactive oxygenspecies (ROS) by this agent and permanent loss of outer hair cells(Kopke et al., Am. J. Otol., 18, 559-571, 1997). Therefore, new orimproved methods are needed for alleviating ototoxicity.

In recent years, RNA interference (“RNAi”) has exhibited potential foruse in many therapeutic applications. It refers to the process ofsequence-specific post-transcriptional gene silencing in animalsmediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature,391, 806).

The process of RNAi begins by the presence of a long dsRNA in a cell,wherein the dsRNA comprises a sense RNA having a sequence homologous tothe target gene mRNA and antisense RNA having a sequence complementaryto the sense RNA. The presence of dsRNA stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes (Elbashir et al., 2001, Genes Dev., 15, 188). siRNAs in turnstimulate the RNA-induced silencing complex (RISC) by incorporating onestrand of siRNA into the RISC and directing the degradation of thehomologous mRNA target.

The original RNAi, which was discovered in invertebrates and employeddsRNAs with length greater than 30 nucleotides was not effective inmammalian cells. This was found to be due to the fact that long dsRNAs(greater than 30 nucleotides) elicit interferon responses, resulting innonspecific mRNA degradation and inhibition of protein synthesis. Thisproblem was overcome by the finding that smaller double-stranded siRNAswith the length of 20-23 nucleotides do not induce an interferonresponse yet remain potent and specific inhibitors of endogenous geneexpression (Elbashir et al., Nature 411, 494-498, 2001).

In research laboratories, two types of siRNA have been widely used tosuppress exogenous as well as endogenous gene expression: syntheticsiRNA and vector-based siRNA (i.e. in vivo transcribed siRNA). Thevector-based siRNA is usually generated through short hairpin RNA(shRNA). In this system, RNA polymerase III promoters, such as H1promoter and U6 promoter are used to drive transcription of shRNA. TheshRNA transcript consists of a 19- to 29-bp RNA stem, with the twostrands joined by a tightly structured loop. shRNA is processed in thecell into siRNA through the action of the Dicer family of enzymes. Thus,the transcribed products mimic the synthetic siRNA duplexes and are aseffective as the synthetic siRNA for suppressing their correspondinggenes.

SUMMARY OF THE INVENTION

It is one embodiment of the present invention to provide a method forpreventing and/or reducing ototoxicity in a patient suffering from or atrisk from developing ototoxicity caused by an ototoxic agent, noise orhead and/or neck radiation or tinnitus, wherein the method comprisessilencing TRPV1 in a patient.

In another embodiment, the present invention relates to a method forpreventing and/or reducing ototoxicity in a patient suffering from or atrisk from developing ototoxicity caused by an ototoxic agent, noise orhead and/or neck radiation or tinnitus, wherein the method comprisesadministering a siRNA directed against TRPV1 mRNA to the patient. Insome alternative embodiments, a siRNA directed against NOX3 mRNA or acombination of siRNAs directed against TRPV- and NOX3 can beadministered. In one preferred embodiment, a sense strand of the siRNAdirected against TRPV1 has a sequence of SEQ ID NO: 1. In anotherpreferred embodiment, a sense strand of the siRNA against TRPV1 has asequence of SEQ ID NO: 2. In still another preferred embodiment, a sensestrand of the siRNA directed against NOX3 has a sequence of SEQ ID NO:3. In yet another preferred embodiment, the siRNA of the presentinvention is administered locally.

It is yet another embodiment of the present invention to provide amethod for preventing or reducing generation of reactive oxygen speciesin inner ear of a patient, the method comprising administering to thepatient at least one siRNA selected from siRNA directed against TRPV1mRNA and NOX3 mRNA.

Other features of the present invention will be in part apparent and inpart pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the increased TRPV1 expression in organ of Corti oncisplatin treatment. (A) Cochleae from rats treated were harvested after3 days post cisplatin treatment, sectioned and stained with TRPV1antibody. Increased immunoreactivity was observed in cisplatin treatedsamples, compared to control as seen in the outer hair cells (OHC) andspiral ganglion (SG). (B) Increased TRPV1 protein expression was seen inthe rat cochlea 72 hrs post cisplatin treatment. (C) TRPV1 mRNAexpression in the rat cochlea increased as early as 24 hrs postcisplatin treatment, with no further significant change in expressionlevels over 48 and 72 hrs. (D) Lipoic acid pretreatment before cisplatintreatment (72 hrs) inhibited the upregulation of TRPV1 mRNA in ratcochlea suggesting involvement of ROS in the regulation of TRPV1. (E)Increased NOX3 mRNA expression was seen 72 hrs post cisplatin treatmentin the rat cochlea. (F) Increased NOX3 mRNA expression was seen as earlyas 24 hrs post cisplatin treatment in the rat cochlea, with little or nofurther change at 48 and 72 hrs. (G) Lipoic acid pretreatment abolishedthe increase in NOX3 mRNA on cisplatin treatment (72 hrs), implicatingROS as the trigger for NOX3 induction in the cochlea. (*p<0.05 comparedto control, students T test).

FIG. 2 shows increased TRPV1, NOX3, and other NADPH oxidase isoformexpression following cisplatin administration in vitro. (A) IncreasedTRPV1 protein immunoreactivity was seen in UB/OC-1 cells treated with2.5 μM cisplatin within 24 hrs post treatment. NADPH oxidase inhibitorslike DPI and AEBSF downregulated the increase in TRPV1immunoflourescence on cisplatin treatment 2.5 μM in UB/OC-1 cellsimplicating ROS in cisplatin mediated ototoxicity. (B) Graphicalrepresentation of average intensity of immunofluorescence seen in FIG.2(A) is shown. (*p<0.05 compared to control, **p<0.05 compared tocisplatin, students T test). (C) Cisplatin 2.5 μM treatment increasedTRPV1 protein expression significantly over control in UB/OC-1 cells.(D) Graphical representation of TRPV1 protein expression as shown inFIG. 2(C). (E) Three fold increase in TRPV1 mRNA expression was alsoobserved in the UB/OC-1 cells on cisplatin 2.5 μM treatment (24 hrs) byreal time RT-PCR. (F) UB/OC-1 cells on 30 minute cisplatin 2.5 μMtreatment showed a marked increase in ROS generation as determined byDCF2DA dye. This increase in ROS generation was abolished bypretreatment with AEBSF an NADPH oxidase inhibitor, indicating a rolefor NADPH oxidases. (G) Increase in NOX3, gp-91 and Rac-1 mRNA was seenin UB/OC-1 cells treated with cisplatin (2.5 μM) that was abolished bypretreatment with DPI, an NADPH oxidase inhibitor. (H) Increased celldeath was seen on 24 hr cisplatin treatment (20 μM) as seen by TUNEL.(I) Pretreatment with capsazepine and ruthenium red, antagonists ofTRPV1 reversed the trend seen in FIG. 2(H) significantly. (*p<0.05compared to control, **p<0.05 compared to control, student's T test). (Jand K) Increased expression of pro-apoptotic proteins like p53 and Baxwas seen in UB/OC-1 cells exposed to 24 hr cisplatin treatment overcontrol cells. Pre-treatment with TRPV1 antagonist like capsazepine andcalcium chelator like BAPTA-AM before cisplatin administrationdown-regulated the expression of these pro-apoptotic genes to controllevels.

FIG. 3 depicts protective effects of TRPV1 siRNA directed againstcisplatin induced ototoxicity in rat model. (A) UB/OC-1 cells weretransfected with siNOX3 and siTRPV1 for 24 hrs and then treated withcisplatin 2.5 μM for another 24 hrs. NOX3 mRNA expression was reducedunder basal levels as well as in cisplatin treated samples transfectedwith siNOX3. siTRPV1 did not change basal expression of NOX3 mRNA butdid abolish the increase in NOX3 mRNA on subsequent cisplatin treatment.TRPV1 mRNA expression showed a similar pattern, suggesting co-regulationof these proteins in cisplatin induced ototoxicity. (B) PretreatmentABRs were measured in rat models, followed by round window applicationof siTRPV1 (0.9 μg per ear) for 48 hrs, cisplatin administration (13mg/kg i.p.) for 72 hrs and post ABRs were then collected. A significantdecrease in threshold shift was observed at both 8 and 16 KHz in thesiTRPV1 treated animals compared to the vehicle treated control animalsimplying otoprotection with siTRPV1. (C) TRPV1 mRNA expression insiTRPV1 treated cochlear samples showed an 85% down regulation comparedto control cochlea. (D) Graphic representation of TRPV1 protein levelsin the cochlea harvested from the siTRPV1 treated animals showedsignificant down regulation of TRPV1 compared to control. Pretreatmentwith siTRPV1 prior to cisplatin administration also showed significantdecrease in TRPV1 expression compared to cisplatin treatment alone.(*p<0.05 compared to control, **p<0.05 compared to cisplatin, students Ttest). (E) Western blot of TRPV1 expression as described in FIG. 3(D).(F) Scanning electron microscopy images of the hook, basal turn and themiddle turn of the cochleae of rats pretreated with either PBS orsiTRPV1 48 hrs prior to cisplatin administration (72 hrs) showedsignificant protection in hair cell damage in all the turns. (G) showsthe semi-quantitative graphical analysis of the percentage of hair celldeath in all the samples from FIG. 3(F). Significant hair cellprotection is seen in siTRPV1 treated cochleae compared to PBS treatedcontrols at all the turns. (*p0.05 compared to PBS treated control,student's t-test). (H) is a diagram of a proposed mechanism forcisplatin-induced hair cell apoptosis.

FIG. 4 shows that noise exposure increases the expression of TRPV1 andNADPH oxidase subunits in the rat cochlea. (A) Rats were exposed toambient noise (˜60 dB) (shaded bars) or 90 dB noise (open bars) for 8 hand auditory brainstem evoked responses (ABRs) measures were performedusing test frequencies of 8 and 16 kHz. The 90 dB noise exposure induceda transient shift in ABR thresholds by ˜40 dB, compared to the pre-noiseABR (*, statistically significant difference, p<0.05). The 60 dB ambientnoise levels did not produce significant shifts in ABR thresholds. (B)Demonstration of NAD(P)H activity in cochlear lysates using NADH andNADPH as substrates. (C) Noise increased NADPH oxidase activity in therat cochlea at 1 and 3 days. (D) Noise exposure resulted in theinduction of NADPH oxidase subunits, NOX3 and Rac1, along with TRPV1.

FIG. 5 depicts that inhibition of TRPV1 reduces generation of reactiveoxygen species (ROS) by aminoglycosides. UB/OC-1 cultures werepretreated with vehicle (control), capsazepine (10 μM) or ruthenium red(20 μM) for 15 min prior to the administration of vehicle (control) orgentamicin (20 μM) for 30 min and the generation of reactive oxygenspecies was determined by the fluorescence of2′,7′-dichlorodihydrofluorescein diacetate (DCF2DA) using confocalmicroscopy. The left side shows the ROS generation image of thegentamicin group which was suppressed by capsazepine and ruthenium red.The right side shows the Nomarski images of the corresponding cells onthe left.

DEFINITIONS

The term “complementary” refers to the ability of polynucleotides toform base pairs with one another. Base pairs are typically formed byhydrogen bonds between nucleotide units in antiparallel polynucleotidestrands. Complementary polynucleotide strands can base pair in theWatson-Crick manner (e.g., A to T, A to U, C to G), or in any othermanner that allows for the formation of duplexes. As persons skilled inthe art are aware, when using RNA as opposed to DNA, uracil rather thanthymine is the base that is considered to be complementary to adenine.However, when a U is denoted in the context of the present invention,the ability to substitute a T is implied, unless otherwise stated.Perfect complementarity or 100% complementarity refers to the situationin which each nucleotide unit of one polynucleotide strand can hydrogenbond with a nucleotide unit of a second polynucleotide strand. Less thanperfect complementarity refers to the situation in which some, but notall, nucleotide units of two strands can hydrogen bond with each other.For example, for two 20-mers, if only two base pairs on each strand canhydrogen bond with each other, the polynucleotide strands exhibit 10%complementarity. In the same example, if 18 base pairs on each strandcan hydrogen bond with each other, the polynucleotide strands exhibit90% complementarity.

The term “expression cassette” is used to define a nucleotide sequencecontaining regulatory elements operably linked to a coding sequence thatresult in the transcription and translation of the coding sequence in acell.

The term “expression vector” refers to both viral and non-viral vectorscomprising a nucleic acid expression cassette.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor or RNA (e.g., tRNA, siRNA, rRNA, etc.). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction, etc.) ofthe full-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the sequences located adjacent tothe coding region on both the 5′ and 3′ ends, such that the genecorresponds to the length of the full-length mRNA. The sequences thatare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ untranslated sequences. The sequences that arelocated 3′ or downstream of the coding region and that are present onthe mRNA are referred to as 3′ untranslated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region, which may be interruptedwith non-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are removed or “spliced out” from thenuclear or primary transcript, and are therefore absent in the messengerRNA (mRNA) transcript. The mRNA functions during translation to specifythe sequence or order of amino acids in a nascent polypeptide.

The terms “homology” or “homologous” when used in the context of nucleicacid or polypeptide sequences refer to sequence identity or similaritybetween two or more sequences. The degree of sequence identity isgenerally quantified using percentages, which is calculated based on thenumber of differing nucleotides or amino acids over the total length ofthe sequence. As a practical matter, whether any particular nucleic acidmolecule is at least, e.g., 70%, 80%, 90%, 95%, 96%, 97% 98%, 99% or100% identical to the ribonucleotide sequence of a target agent or viruscan be determined conventionally using known computer programs such asthe Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, Madison, Wis.). Bestfit uses the localhomology algorithm of Smith and Waterman (Advances in AppliedMathematics 2:482-489 (1981)) to find the best segment of homologybetween two sequences. When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the referenceribonucleotide sequence and that gaps in homology of up to 5% of thetotal number of ribonucleotides in the reference sequence are allowed.

A “mammalian promoter” refers to a transcriptional promoter thatfunctions in a mammalian cell that is derived from a mammalian cell, orboth.

“NADPH” is an abbreviation for nicotinamide adenine dinucleotidephosphate.

“NOX3” is an abbreviation for NADPH oxidase 3.

By “ototoxic agent” in the context of the present invention is meant asubstance that through its chemical action injures, impairs, or inhibitsthe activity of a cell or tissue component related to hearing, which inturn impairs hearing and/or balance. In the context of the presentinvention, ototoxicity includes a deleterious effect on the inner earsensory hair cells. Ototoxic agents that cause hearing impairmentsinclude, but are not limited to therapeutic drugs includingantineoplastic agents such as vincristine, vinblastine, cisplatin,taxol, or dideoxy-compounds, e.g., dideoxyinosine; salicylates;quinines; diuretics including furosemide and ethocrynic acid;aminoglycosides; polypeptide antibiotics; contaminants in foods ormedicinals; environmental or industrial pollutants; solvents includingtoluene, xylene, metalloproteins including arsenic and cadmium, andlarge doses of vitamins such as vitamins A, D, or B6. By “exposure to anototoxic agent” is meant that the ototoxic agent is made available to,or comes into contact with a patient, such as a human. Exposure to anototoxic agent can occur by direct administration, e.g., by ingestion oradministration of a food, medicinal, or therapeutic agent, e.g., achemotherapeutic agent, by accidental contamination, or by environmentalexposure, e.g., aerial or aqueous exposure.

The term “patient”, as used herein, refers to an animal, preferably amammal. More preferably the patient can be a primate, includingnon-humans and humans. The terms subject and patient are usedinterchangeably herein.

“Pharmaceutically acceptable carrier” includes, but is not limited to, anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type, such as liposomes.

The term “plasmid” as used herein, refers to an independentlyreplicating piece of DNA. It is typically circular and double-stranded.

“Small interfering RNA” (siRNA) refers to double-stranded RNA moleculesfrom about 10 to about 30 nucleotides long that are named for theirability to specifically interfere with protein expression. The length ofthe siRNA molecule is based on the length of the antisense strand of thesiRNA molecule.

“Transfection” is the term used to describe the introduction of foreignmaterial such as foreign DNA into eukaryotic cells. It is usedinterchangeably with “transformation” and “transduction” although thelatter term, in its narrower scope refers to the process of introducingDNA into cells by viruses, which act as carriers. Thus, the cells thatundergo transfection are referred to as “transfected,” “transformed” or“transduced” cells.

A “therapeutically effective” amount of the inventive compositions canbe determined by prevention or amelioration of adverse conditions orsymptoms of diseases, injuries or disorders being treated.

“TRPV1” is an abbreviation for transient receptor potential vanilloidtype 1. It is also known as vanilloid receptor type 1.

As used herein, a “3′ overhang” refers to at least one unpairednucleotide extending from the 3′-end of a duplexed RNA strand.

The term “vector” refers to a DNA molecule into which foreign fragmentsof DNA may be inserted. Generally, they contain regulatory and codingsequences of interest. The term vector includes but is not limited toplasmids, cosmids, phagemids, viral vectors and shuttle vectors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for reducing or preventingototoxicity caused by an ototoxic agent, noise, head and/or neckradiation, or tinnitus. In one embodiment, the method of the presentinvention involves silencing the expression of TRPV1 gene in the ear ofa patient, preferably by administration of siRNA directed against TRPV1mRNA. In another embodiment, the method involves administration of siRNAdirected against NOX3 mRNA. In still another embodiment, the method ofthe present invention involves administration of at least two siRNAsdirected against TRPV1 mRNA and NOX3 mRNA. Furthermore, the presentinvention also relates to a method for preventing or reducing generationof reactive oxygen species in an inner ear of a patient by administeringat least one siRNA selected from siRNA directed against TRPV1 mRNA andNOX3 mRNA.

In one embodiment, the ototoxic agent is selected from aminoglycosidesand platinum-containing chemotherapeutic agents. Aminoglycosides includebut are not limited to neomycin, paromomycin, ribostamycin, lividomycin,kanamycin, amikacin, tobramycin, viomycin, gentamicin, sisomicin,netilmicin, streptomycin, dibekacin, fortimicin, anddihydrostreptomycin, or combinations thereof. In preferred embodiments,the aminoglycosides are selected from neomycin, kanamycin, gentamicin,tobramycin and streptomycin. In still other preferred embodiments, theaminoglycosides are selected from neomycin B, kanamycin A, kanamycin B,gentamicin C1, gentamicin C1a, and gentamicin C2.

Platinum-containing chemotherapeutic agents include but are not limitedto cisplatin and carboplatin. In one preferred embodiment, theplatinum-containing chemotherapeutic agent is cisplatin.

Head and/or neck radiation is generally administered for purposes oftreating head and/or neck cancers including but not limited to tumors ofthe oral cavity, larynx, pharynx, and major salivary glands. In apreferred embodiment, head and/or neck radiation is administered forpurposes of treating squamous cell carcinoma of the oral cavity, larynxor pharynx.

Hearing Loss Evaluation

Tests are known and available for diagnosing hearing impairments.Neuro-otological, neuro-ophthalmological, neurological examinations, andelectro-oculography can be used. (Wennmo et al. Acta Otolaryngol (1982)94:507-15). Sensitive and specific measures are available to identifypatients with auditory impairments. For example, tuning fork tests canbe used to differentiate a conductive from a sensorineural hearing lossand determine whether the loss is unilateral. An audiometer is used toquantify hearing loss, measured in decibels. With this device thehearing for each ear is measured, typically from 125 to 8000 Hz, andplotted. The speech recognition threshold, the intensity at which speechis recognized as a meaningful symbol, can be determined at variousspeech frequencies. Speech or phoneme discrimination can also bedetermined and used as an indicator of sensorineural hearing loss sinceanalysis of speech sounds relies upon the inner ear and the 8^(th)nerve.

Tympanometry can be used to diagnose conductive hearing loss and aid inthe diagnosis of those patients with sensorineural hearing loss.

Electrocochleography (i.e., measuring the cochlear microphonic responseand action potential of the 8^(th) nerve to acoustic stimuli), andevoked response audiometry (i.e., measuring evoked response from thebrainstem to acoustic stimuli) can be used in patients, particularlyinfants and children or patients with sensorineural hearing loss ofobscure etiology. These tests serve a diagnostic function as well as aclinical function in assessing response to therapy.

Sensory and neural hearing losses can be distinguished based on testsfor recruitment (an abnormal increase in the perception of loudness orthe ability to hear loud sounds normally despite a hearing loss),sensitivity to small increments in intensity, and pathologic adaptation,including neural hearing loss. In sensory hearing loss, the sensation ofloudness in the affected ear increases more with each increment inintensity than it does in the normal ear. Sensitivity to smallincrements in intensity can be demonstrated by presenting a continuoustone of 20 dB above the hearing threshold and increasing the intensityby 1 dB briefly and intermittently. The percentage of small incrementsdetected yields the “short increment sensitivity index” value. Highvalues, 80 to 100%, are characteristic of sensory hearing loss, whereasa neural lesion patient and those with normal hearing cannot detect suchsmall changes in intensity. Pathologic adaptation is demonstrated when apatient cannot continue to perceive a constant tone above threshold ofhearing, also known as tone decay. A Bekesy automatic audiometer orequivalent can be used to determine these clinical and diagnostic signs;audiogram patterns of the Type II pattern, Type III pattern and Type IVpattern are indicative of preferred hearing losses suitable for thetreatment methods of the invention. As hearing loss can often beaccompanied by vestibular impairment, vestibular function can be tested,particularly when presented with a sensorineural hearing loss of unknownetiology.

When possible, diagnostics for hearing loss, such as audiometric tests,should be performed prior to exposure in order to obtain a patient'snormal hearing baseline. Upon exposure, particularly to an ototoxic drugor head and/or neck radiation, audiometric tests can be performed, e.g.,twice a week and testing should be continued for a period aftercessation of the ototoxic drug treatment or head and neck radiation,since hearing loss may not occur until several days after cessation. Forexample, U.S. Pat. Nos. 5,546,956 and 4,637,402 provide methods fortesting hearing and measuring hearing defects that can be used todiagnose the patient and monitor treatment.

siRNA

In some of the embodiments of the present invention, TRPV1 geneexpression in the ear is silenced in order to reduce or preventototoxicity caused by an ototoxic agent, noise, head and/or neckradiation or tinnitus. TRPV1 gene expression can be silenced by usingsiRNA directed against TRPV1, antisense nucleotides directed againstTRPV1, or other methods (e.g., by the use of inhibitors of p38 mitogenactivator protein kinase—MAPK (Puntambekar et al., 2006, J. Neurochem.95:1680-1703; Ji et al., 2003, Neuron 36:57-68), which can prevent orsignificantly reduce TRPV1 mRNA or TRPV1 protein expression. Theantisense technology is well known in the art. Briefly, a nucleotidesequence generally containing between 19 and 29 nucleotides is used,which is complementary to the sense mRNA sequence of TRPV1. The degreeof complementarity generally ranges from about 70% to about 100%.Preferably, complementarity is greater than 80%, more preferably greaterthan 90%, and even more preferably greater than 95%. The region of TRPV1mRNA that should be targeted can be readily determined by comparing theefficacy of several antisense sequences designed to complement differentregions of TRPV1 mRNA to prevent production of TRPV1 protein. Suchexperiments can be readily performed without undue experimentation byany of known techniques in the art, such as Western blotting.

In some embodiments, the silencing of the TRPV1 gene is performed byadministering a siRNA directed against TRPV1, wherein the siRNA ispreferably applied locally to the ear. By way of example, the siRNA canbe administered to the round window or intra-tympanically. In anotherpreferred embodiment, a sense strand of the siRNA has a sequence of SEQID NO: 1. In still another preferred embodiment, a sense strand of thesiRNA against TRPV1 has a sequence of SEQ ID NO: 2. It should be notedthat the sequence of SEQ ID NO: 1 differs from SEQ ID NO: 2 by havingthe first 2 nucleotides at 5′ removed and by containing 2 extranucleotides at 3′. SEQ ID NO: 2 was described in a publication byChristoph et al., Biochemical and Biophysical Research Communications350 (2006) 238-243.

In other embodiments, the present invention relates to a method forpreventing and/or reducing ototoxicity in a patient caused by anototoxic agent, noise, head and/or neck radiation or tinnitus, whereinthe method comprises administering more than one siRNA directed againstTRPV1 mRNA. By way of example, two siRNAs directed against TRPV1 can beadministered, such as a combination of siRNAs whose sense sequencesinclude SEQ ID NO: 1 and SEQ ID NO: 2.

In some other embodiments, the present invention relates to a method forpreventing and/or reducing ototoxicity in a patient caused by anototoxic agent, noise or head and neck radiation, wherein the methodcomprises administering a siRNA directed against NOX3 mRNA to thepatient. In one preferred embodiment, a sense strand of the siRNAdirected against NOX3 mRNA contains the sequence of SEQ ID NO: 3. Thereare also commercially available siRNAs directed against NOX3. By way ofexample, Ambion offers at least four siRNAs directed against NOX3, whichtarget exons 2, 5, 6 or 13. See siRNA ID numbers 23254, 118793, 118794and 23441, respectively.(http://www.ambion.com/catalog/sirna_search.php?action=pre&sirna_id=118794)Preferably, the siRNA against NOX3 is applied locally to the ear of apatient, e.g., by round window administration.

In additional embodiments, the present invention provides a method forpreventing and/or reducing ototoxicity in a patient caused by anototoxic agent, noise, head and/or neck radiation, or tinnitus, whereinthe method comprises administering at least one siRNA selected fromsiRNAs directed against TRPV1 mRNA and NOX3 mRNA. In one preferredembodiment, the method comprises administering a combination of a siRNAdirected against TRPV1 and a siRNA directed against NOX3. In anotherpreferred embodiment, the method comprises administering a combinationof siRNAs whose sense strands include SEQ ID NO: 1 and SEQ ID NO: 3. Instill another preferred embodiment, the combination of at least twosiRNAs comprises siRNAs with sense strands SEQ ID NO: 2 and SEQ ID NO:3. In another preferred embodiment, a combination of siRNAs having sensestrands comprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 isadministered. Preferably, the siRNAs against TRPV1 and NOX3 are appliedlocally to the ear of a patient, e.g., by round window administration orintra-tympanically.

In addition to the above-mentioned ototoxic agents, salicylates (such asasprin and aspirin-containing drugs) and non-steroidal anti-inflammatorydrugs (NSAIDs) (such as naproxen, ibuprofen, diclofenac, piroxicam,indomethacin and the like) can also induce temporary hearing loss.Accordingly, the siRNAs of the present invention can also be used toprevent and/or reduce ototoxicity associated with administration ofthese agents. Preferably, the administration of at least one siRNA ofthe present invention is started prior to the start of the salicylate orNSAID regimen, and is continued during such regimen, if needed. Thus, atleast one siRNA selected from siRNAs directed against TRPV1 and NOX3 canbe used to treat and/or prevent ototoxicity associated with theadministration of NSAIDs or salicylates.

As known in the art, the siRNA contains an antisense strand and a sensestrand which form an RNA duplex. Thus, the siRNAs of the presentinvention contain either the TRPV1 antisense and sense sequences or NOX3antisense and sense sequences.

Thus, in one embodiment of the invention, the antisense RNA sequence isat least 70% complementary to an RNA sequenced from either TRPV1 orNOX3. In other preferred embodiments, the TRPV1 and NOX3 antisensesequences are at least, 80, 90, 95, or 100% complementary to TRPV1 mRNAand NOX3 mRNA sequences, respectively. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment or, in the case of in vitro assays,under conditions in which the assays are conducted.

One skilled in the art can readily determine substitutions and/ormutations in antisense RNA which result in such complementarity. By wayof example, if the NOX3 mRNA sequence (sense) and antisense sequencesare each 20 nucleotides long, the antisense sequence is 90%complementary to the sense mRNA sequence if 18 out of the 20 nucleotidesin the antisense strand can base pair with the nucleotides in the NOX3mRNA. By way of another example, if the TRPV1 mRNA sequence (sense) andantisense sequences are each 25 nucleotides long, the antisense sequenceis 80% complementary to the sense mRNA sequence if 20 out of the 25nucleotides in the antisense strand can base pair with the nucleotidesin the TRPV1 mRNA. When making such substitutions, considerations suchas where they are introduced and whether they are dispersed throughoutthe sequence or occur together can affect the efficacy of the siRNA. Byway of example, it is known in the art that substitutions in the centerof the molecule tend to affect the efficacy to a greater degree than thesubstitutions at either end of the molecule. Similarly, two or morecontiguous substitutions tend to affect the ability of the antisense RNAto bind to a target molecule to a greater degree than two or moremutations situated throughout the antisense sequence. In one embodiment,the substitutions are introduced such that there are regions of at least3, more preferably of at least 4, and even more preferably of at least 5contiguous unmutated nucleotides between each substitution.

The following features are not required but can also be considered whendetermining which substitutions to make: (1) G/C content of about 25% toabout 60% G/C; (2) at least 3 A/Us at positions 15-19 of the sensestrand; (3) no internal repeats; (4) an A at position 19 of the sensestrand; (5) an A at position 3 of the sense strand; (6) a U at position10 of the sense strand; (7) no G/C at position 19 of the sense strand;and (8) no G at position 13 of the sense strand.

In addition, if a substitution results in a potential siRNA targetsequence with one or more of the following criteria, such sequence maybe less likely to function as siRNA: (1) sequence comprising a stretchof 4 or more of the same base in a row; (2) sequence comprisinghomopolymers of Gs; (3) sequence comprising triple base motifs (e.g.,GGG, CCC, AAA, or TTT); (4) sequence comprising stretches of 7 or moreG/Cs in a row; and (5) sequence comprising direct repeats of 4 or morebases within the candidates resulting in internal fold-back structures.However, such sequences should still be evaluated for the ability tofunction as siRNA molecules.

Accordingly, one of ordinary skill in the art can determine withoutundue experimentation which substitutions to make in order to achievethe desired complementarity between the sense RNA and antisense RNA.

In addition to substitutions, one or more nucleotides can be chemicallymodified, e.g., for purposes of reducing immunostimulatory effect ofsiRNA sequences. In some embodiments, the chemically modified siRNAcomprises modified nucleotides including, but not limited to,2′-O-methyl (2′OMe) nucleotides, 2′-deoxy-2′-fluoro (2′F) nucleotides,2′-deoxy nucleotides, 2′-O-(2-methoxyethyl) (MOE) nucleotides, lockednucleic acid (LNA) nucleotides, and mixtures thereof. In preferredembodiments, the modified siRNA comprises 2′OMe nucleotides (e.g., 2′OMepurine and/or pyrimidine nucleotides) such as, for example,2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, 2′OMe-adenosinenucleotides, 2′OMe-cytosine nucleotides, and mixtures thereof. Themodified siRNA can comprise modified nucleotides in one strand (i.e.,sense or antisense) or both strands of the double-stranded region of thesiRNA.

In one embodiment, the antisense RNA of the siRNA directed against TRPV1is from about 19 to about 29 nucleotides long. In another embodiment,the antisense RNA of the siRNA directed against NOX3 is from about 19 toabout 29 nucleotides long. Preferably, the antisense RNA of the siRNAmolecules of the present invention, namely TRPV1 siRNA or NOX3 siRNA is20-28 nucleotides long, and still more preferably 21-25 nucleotideslong. Therefore, preferred antisense strands of siRNA molecules are 19,20, 21, 22, 23, 24, 25, 26, 27 28 or 29 nucleotides in length. The sensestrand of the siRNA of the present invention is also from about 19 toabout 29 nucleotides long, preferably 20-28 nucleotides long, and stillmore preferably 21-25 nucleotides long. Therefore, preferred sensestrands of siRNA molecules of the present invention are 19, 20, 21, 22,23, 24, 25, 26, 27 28 or 29 nucleotides in length. In another preferredembodiment, the antisense strand is of the same length as the sensestrand.

In one embodiment, the sense and antisense strands of the present siRNAare composed of two complementary, single-stranded RNA molecules. Inanother embodiment, the sense and antisense strands are encoded by asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area. Withoutwishing to be bound by any theory, it is believed that the hairpin areaof the latter type of siRNA molecule is cleaved intracellularly by the“Dicer” protein (or its equivalent) to form an siRNA of two individualbase-paired RNA molecules (see Tuschl, T. (2002), supra). Thus, siRNAsagainst TRPV1 and NOX3 can either be composed of two single-stranded RNAmolecules or can be composed of a single molecule in which the sense andantisense strands are separated by a hairpin.

The siRNA of the present invention can comprise partially purified RNA,substantially pure RNA, synthetic RNA, or recombinantly produced RNA, aswell as altered RNA that differs from naturally-occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA, including modifications that make the siRNAresistant to nuclease digestion. Amino acid deletions, substitutions oradditions can be carried out by a site-specific mutagenesis method whichis a well known technique. See, for example, Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989); Current Protocols in Molecular Biology, Supplement 1 to 38, JohnWiley & Sons (1987-1997); Nucleic Acids Research, 10, 6487 (1982); Proc.Natl. Acad. Sci., USA, 79, 6409 (1982); Gene, 34, 315 (1985); NucleicAcids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci. USA, 82, 488(1985); Proc. Natl. Acad. Sci., USA, 81, 5662 (1984); Science, 224, 1431(1984); PCT WO85/00817 (1985); Nature, 316, 601 (1985); and the like.

One or both strands of the siRNA of the invention can also comprise a 3′overhang. Thus in some embodiments, the siRNA directed against TRPV1 orNOX3 comprises at least one 3′ overhang of from 1 to about 6 nucleotides(which includes ribonucleotides or deoxynucleotides) in length,preferably from 1 to about 5 nucleotides in length, more preferably from1 to about 4 nucleotides in length, and even more preferably from about2 to about 4 nucleotides in length.

In the embodiment in which both strands of the siRNA molecule comprise a3′ overhang, the length of the overhangs can be the same or differentfor each strand. In a most preferred embodiment, the 3′ overhang ispresent on both strands of the siRNA, and is 2 nucleotides in length.For example, each strand of the siRNA of the invention can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

In order to enhance the stability of the present siRNA, the 3′ overhangscan be also stabilized against degradation. In one embodiment, theoverhangs are stabilized by including purine nucleotides, such asadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, istolerated and does not affect the efficiency of RNAi degradation. Inparticular, the absence of a 2′ hydroxyl in the 2′-deoxythymidinesignificantly enhances the nuclease resistance of the 3′ overhang intissue culture medium.

siRNA Preparation

siRNA can be prepared in a number of ways, such as by chemicalsynthesis, T7 polymerase transcription, or by treating long doublestranded RNA (dsRNA) prepared by one of the two previous methods withDicer enzyme. Dicer enzyme creates mixed populations of dsRNA from about21 to about 23 base pairs in length from dsRNA that is about 500 basepairs to about 1000 base pairs in size. Dicer can also effectivelycleave modified strands of dsRNA, such as 2′ fluoro-modified dsRNA. TheDicer method of preparing siRNAs can be performed using a Dicer siRNAGeneration Kit available from Gene Therapy Systems (San Diego, Calif.).

In one preferred embodiment, the siRNA directed against TRPV1 or NOX3 issynthetically produced. By way of example and not of limitation, thesiRNAs of the present invention are chemically synthesized usingappropriately protected ribonucleotides phosphoramidites and aconventional DNA/RNA synthesizer. The siRNA can be synthesized as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo. USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va. USA),ChemGenes (Ashland, Mass. USA) and Cruachem (Glasgow, UK). The siRNA ofthe present invention can be a recombinantly produced RNA. A number ofsiRNAs are commercially available and can be purchased from vendors suchas Ambion and Santa Cruz Biotechnology, Inc.

A variety of different vectors can be employed for producing siRNAs byrecombinant techniques. Such vectors include chromosomal, nonchromosomaland synthetic DNA sequences, e.g., derivatives of SV40; bacterialplasmids; phage DNA; baculovirus; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, and pseudorabies. However, any other vectorcan be used as long as it is replicable and viable in a desired host.

The siRNA of the present invention can be expressed from a recombinantplasmid either as two separate, complementary RNA molecules, or as asingle RNA molecule with two complementary regions.

Selection of plasmids suitable for expressing siRNA of the invention,methods for inserting nucleic acid sequences for expressing the siRNAinto the plasmid, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Tuschl, T. (2002), Nat. Biotechnol, 20: 446-448, Brummelkamp T R et al.(2002), Science 296: 550-553 Miyagishi M et al, (2002), Nat. Biotechnol.20: 497-500; Paddison P J et al. (2002). Genes Dev. 16: 948-9578; Lee NS et al. (2002) Nat. Biotechnol, 20: 500-505; and Paul C P et al.(2002). Nat. Biotechnol. 20: 505-508. Selection of viral vectorssuitable for use in the present invention are also within the skill inthe art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310;Eglitis M A (1988), Biotechniques 6: 608-1614; Miller A D (1990), HumGene Therap. 1: 5-14; and Anderson W F (1998), Nature 392: 25-30.

The appropriate DNA segment may be inserted into the vector by a varietyof procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart, which can be performed without undue experimentation by a skilledartisan. The DNA segment in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directsiRNA synthesis. Suitable eukaryotic promoters include the CMV immediateearly promoter, the herpes simplex virus (HSV) thymidine kinasepromoter, the early and late SV40 promoters, the promoters of retrovirallong terminal repeats (LTRs), such as those of the Rous Sarcoma Virus(RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter. Preferably, the promoters of the presentinvention are from the type III class of RNA polymerase III promoters.More preferably, the promoters are selected from the group consisting ofthe U6 and H1 promoters. In still another preferred embodiment, thepromoter is a U6 promoter.

The promoters of the present invention may also be inducible, in thatexpression may be turned “on” or “off.” For example, atetracycline-regulatable system employing the U6 promoter may be used tocontrol the production of siRNA. Additionally, promoters which aretissue specific or respond to a particular stimulus can also be used. Byway of example and not of limitation, tissue specific promoters includepromoters which are active in the liver, such as, e.g., albuminpromoter. Promoters which respond do a particular stimulus include,e.g., heat shock protein promoters, and Tet-off and Tet-on promoters.

In addition, the expression vectors preferably contain one or moreselectable marker genes to, provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or tetracycline or ampicillinresistance.

In one embodiment, the invention provides a vector, wherein the DNAsegment encoding the sense strand of the RNA polynucleotide is operablylinked to a first promoter and where the DNA segment encoding theantisense (opposite) strand of the RNA polynucleotide molecule isoperably linked to a second promoter. In other words, each strand of theRNA polynucleotide is independently expressed. Furthermore, the promoterdriving expression of each strand can be identical or each one may bedifferent from the other promoter. In another embodiment, the vectorused to express a siRNA of the present invention can include opposingpromoters. For example, the vector can contain two U6 promoters oneither side of the DNA segment encoding the sense strand of the RNApolynucleotide and placed in opposing orientations, with or without atranscription terminator placed between the two opposing promoters. TheU6 opposing promoter construct is similar to the T7 opposing promoterconstruct as described in, e.g., Wang, Z. et al., J. Biol. Chem. 275:40174-40179 (2000). In another embodiment, the DNA segments encodingboth strands of the RNA polynucleotide are under the control of a singlepromoter. In one embodiment, the DNA segments encoding each strand arearranged on the vector with a “loop” region interspersed between the twoDNA segments, where transcription of the DNA segments and loop regioncreates one RNA transcript. The single transcript will, in turn, annealto itself creating a “hairpin” RNA structure capable of inducing RNAi.The “loop” of the hairpin structure is preferably from about 4 to about12 nucleotides in length. More preferably, the loop is 9 nucleotides inlength.

Any viral vector capable of accepting the coding sequences for the siRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.lentiviruses (LV), Rhabdoviruses; herpes virus, and the like. Thetropism of the viral vectors can also be modified by pseudotyping thevectors with envelope proteins or other surface antigens from otherviruses. For example, an AAV vector of the invention can be pseudotypedwith surface proteins from vesicular stomatitis virus (VSV), rabies,Ebola, Mokola, and the like. In one embodiment, preferred viral vectorsare those derived from lentiviruses.

The vector containing the appropriate DNA sequence as described herein,as well as an appropriate promoter or control sequence, can be employedto transform an appropriate host to permit the host to express thesiRNA. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989). In one embodiment, the cells used to produce siRNAsof the present invention are HEK 293T cells.

Detection of siRNA

The ability of an siRNA containing a given target sequence to causeRNA-mediated degradation of the target mRNA can be evaluated usingstandard techniques for measuring the levels of RNA or protein in cells.Methods for the determination of mRNA expression levels are known in theart and comprise Real Time PCR, Northern blotting and hybridization onmicroarrays or DNA chips.

Additionally, the methods described in the above sections related tohearing tests can also be used to assess the efficacy of siRNAs toprevent or reduce hearing loss.

siRNA Delivery

In the present methods, the present siRNA can be administered to thesubject either as naked siRNA, in conjunction with a delivery reagent,or as a recombinant plasmid or viral vector which expresses the siRNA.

In some preferred embodiments, at least one siRNA of the presentinvention is administered locally to the ear, e.g., by application tothe round window. Delivery of therapeutic agents in a controlled andeffective manner with respect to tissue structures of the inner ear, forexample, those portions of the ear contained within the temporal bonewhich is the most dense bone tissue in the human body, is known in theart. Exemplary inner ear tissue structures of primary importance includebut are not limited to the cochlea, the endolymphatic sac/duct, thevestibular labyrinth, and all of the compartments which include thesecomponents. Access to the foregoing inner ear tissue regions istypically achieved through a variety of structures, including but notlimited to the round window membrane, oval window/stapes footplate, theannular ligament, and systemically. In some preferred embodiments, thesiRNA directed against TRPV1, the siRNA directed against NOX3 or acombination thereof is administered locally to the ear by administrationto the round window membrane.

The siRNA is applied to the round window membrane by one of severalmethods, such as: 1) direct application of the siRNA, in a solution orotherwise, by surgical exposure of the round window membrane andadministering the siRNA with a syringe and blunt needle, or 2) with awick or catheter to direct the siRNA to the round window membrane byusing an endoscope or a surgical microscope to guide the application. Byway of example, the siRNA of the present invention is added as a drop offluid on the round window using a syringe, following which it diffusesslowly into the perilymph over a 1 hour period.

Intratympanic administration can be performed by injection through anintact tympanic membrane or through a ventilation tube surgicallyinserted through the tympanic membrane; or as an otic drop solution,applied to the ear canal and allowed to enter the middle ear through anexisting perforation of the eardrum or through a ventilation tube placedin the tympanic membrane. The entry of the solution into the middle earwhich allows access to the round window membrane is assisted by aprocedure called “tragal pumping” which simply involves pushing thetragus of the ear gently in a medial direction to push the solutionthrough a ventilation tube into the middle ear. The tragus is theportion of the external, ear in front of the ear canal which consists ofskin-covered cartilage. This procedure is routinely used to administerear drops for middle ear infections in patients with a ventilation tubein place or a perforation of the ear drum. By way of example, repeatedapplications of the siRNA can be carried out with a surgically implantedcatheter placed under local anesthesia.

When it is administered as naked siRNA, the delivery to the cells can beachieved, e.g., by electroporation or gene gun method. Both arewell-known in the art, and described, e.g., in Sambrook and Russell,Molecular Cloning: A Laboratory manual, third edition, Cold SpringHarbor Laboratory Press America, 2001.

Suitable delivery reagents for administration in conjunction with thepresent siRNA include the Mirus Transit TKO lipophilic reagent;lipofectin; lipofectamine; cellfectin; or polycations (e.g.,polylysine), or liposomes. A preferred delivery reagent is a liposome.

Liposomes can aid in the delivery of the siRNA to a particular tissue,and can also increase the blood half-life of the siRNA. Liposomessuitable for use in the invention are formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors such as thedesired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9: 467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369. In one embodiment, the liposomes encapsulating the presentsiRNA comprises a ligand molecule that can target the liposome to theear.

The liposomes encapsulating the present siRNA can be modified so as toavoid clearance by the mononuclear macrophage and reticuloendothelialsystems, for example by having opsonization-inhibition moieties bound tothe surface of the structure. In one embodiment a liposome of theinvention can comprise both opsonization-inhibition moieties and aligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer which significantly decreases the uptakeof the liposomes by the macrophage-monocyte system (“MMS”) andreticuloendothelial system (“RES”); e.g., as described in U.S. Pat. No.4,920,016. Liposomes modified with opsonization-inhibition moieties thusremain in the circulation much longer than unmodified liposomes. Forthis reason, such liposomes are sometimes called “stealth” liposomes.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate synthetic polymers such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g.,polyvinylalchohol and polyxylitol to which carboxylic or amino groupsare chemically linked, as well as gangliosides, such as ganglioside GM1.

Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof,are also suitable. In addition, the opsonization inhibiting polymer canbe a block copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. The opsonizationinhibiting polymers can also be natural polysaccharides containing aminoacids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic,acid, carageenan; aminated polysaccharides or oligosaccharides (linearor branched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups.

Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes.”

The opsonization inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination using NaCN)BH3and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratioat 60° C.

Recombinant plasmids which express siRNA of the invention are discussedabove. Such recombinant plasmids can also be administered directly or inconjunction with a suitable delivery reagent, including the MirusTransit LT1 lipophilic reagent; lipofetin; lipofectamine; cellfectin;polycations (e.g., polylysine) or liposomes. Recombinant viral vectorswhich express siRNA of the invention are also discussed above, andmethods for delivering such vectors to the ear of a patient are withinthe skill in the art.

Suitable enteral administration routes include oral, sublingual, rectalor intranasal delivery.

Suitable parenteral administration routes include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intraarterial bolus injection, intraarterial infusion and catheterinstillation into the vasculature); peri- and intra-tissueadministration; subcutaneous injection or deposition includingsubcutaneous infusion (such as by osmotic pumps); and inhalation. In apreferred embodiment, injections or infusions of the siRNA are given inthe ear or near it.

Pharmaceutical Compositions

The siRNA of the invention can be formulated as pharmaceuticalcompositions prior to administering to a subject, according totechniques known in the art. Pharmaceutical compositions of the presentinvention are characterized as being at least sterile and pyrogen-free.Methods for preparing pharmaceutical compositions of the invention arewithin the skill in the art, for example as described in Remington'sPharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa.,(1985).

The present pharmaceutical formulations comprise at least one siRNA ofthe invention (e.g., 0.1 to 90% by weight), or a physiologicallyacceptable salt thereof, mixed with a pharmaceutically acceptablecarrier. Preferred physiologically acceptable carriers are water,buffered water, saline solutions (e.g., normal saline or balanced salinesolutions such as Hank's or Earle's balanced salt solutions), 0.4%saline, 0.3% glycine, hyaluronic acid and the like. In some embodiments,the pharmaceutical composition comprises a siRNA directed against TRPV1.In one embodiment, a sense strand of the siRNA comprises SEQ ID NO: 1.In another embodiment, a sense strand of the siRNA directed againstTRPV1 comprises SEQ ID NO: 2. In another embodiment, the pharmaceuticalcomposition of the present invention comprises at least two siRNAdirected against TRPV1, such as, e.g., at least two siRNAs with sensestrands comprising SEQ ID NO: 1 and SEQ ID NO: 2. The pharmaceuticalcomposition of the present invention can also include at least one siRNAdirected against NOX3. In one embodiment, such pharmaceuticalcomposition can include, e.g., a siRNA with a sense strand comprisingSEQ ID NO: 3. In other embodiments, the pharmaceutical composition ofthe present invention can include a combination of siRNAs directedagainst TRPV1 mRNA and NOX3 mRNA, wherein the sense strands are, e.g.,SEQ ID NO: 1 and SEQ ID NO: 3, and SEQ ID NO: 2 and SEQ ID NO: 3, or SEQID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

The pharmaceutical composition of the present invention can beadministered orally, nasally, parenterally, intrasystemically,intraperitoneally, topically (as by drops or transdermal patch),bucally, sublingually or as an oral or nasal spray. In one preferredembodiment, the pharmaceutical composition of the present invention isadministered locally, such as topically. In still another preferredembodiment, the pharmaceutical composition is given by round windowadministration.

A pharmaceutical composition of the present invention for parenteralinjection can comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

In some cases, to prolong the effect of the drugs, it is desirable toslow the absorption from subcutaneous or intramuscular injection. Thiscan be accomplished by the use of a liquid suspension of crystalline oramorphous material with poor water solubility. The rate of absorption ofthe drug then depends upon its rate of dissolution which, in turn, candepend upon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally administered drug form is accomplished bydissolving or suspending the drug in an oil vehicle. Prolongedabsorption of the injectable pharmaceutical form can be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

Solid dosage forms for oral administration include, but are not limitedto, capsules; tablets, pills, powders, and granules. In such soliddosage forms, the active compounds are mixed with at least one itempharmaceutically acceptable excipient or carrier such as sodium citrateor dicalcium phosphate and/or a) fillers or extenders such as starches,lactose, sucrose, glucose, mannitol, and silicic acid, b) binders suchas, for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, acetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form can also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers insoft and hard filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They can optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

The pharmaceutical compositions of the present invention can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, solutions, suspensions,syrups and elixirs. In addition to the active compounds, the liquiddosage forms can contain inert diluents commonly used in the art suchas, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethyl formamide., oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the siRNAs of the present invention, cancontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,and mixtures thereof. Alternatively, the composition can be pressurizedand contain a compressed gas, such as nitrogen or a liquefied gaspropellant. The liquefied propellant medium and indeed the totalcomposition are preferably such that the active ingredients do notdissolve therein to any substantial extent. The pressurized compositioncan also contain a surface active agent. The surface active agent can bea liquid or solid non-ionic surface active agent or can be a solidanionic surface active agent. It is preferred to use the solid anionicsurface active agent in the form of a sodium salt.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxicants, osmolalityadjucting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate).

Pharmaceutical compositions comprising the siRNAs of the present caninclude penetration enhancers in order to enhance the alimentarydelivery of the oligonucleotides. Penetration enhancers may beclassified as belonging to one of five broad categories, i.e., fattyacids, bile salts, chelating agents, surfactants and non-surfactants(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991,8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems 1990, 7, 1-33). One or more penetration enhancers from one ormore of these broad categories may be included. Various fatty acids andtheir derivatives which act as penetration enhancers include, forexample, oleic acid, lauric acid, capric acid, myristic acid, palmiticacid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, recinleate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,caprylic acid, arachidonic acid, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- anddi-glycerides and physiologically acceptable salts thereof (i.e.,oleate, laurate, caprate, myristate, palmitate, stearate, linoleate,etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol. 1992 44,651-654).

Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) [Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems 1990, 7, 1-33; Buur et al., J. ControlRel. 1990, 14, 43-51).

Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page92); and perfluorochemical emulsions, such as FC43 (Takahashi et al., J.Pharm. Phamacol. 1988, 40, 252-257).

Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl-and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 1987, 39,621-626).

Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol, sorb acid, and the like.

One of ordinary skill in the art will appreciate that effective amountsof the agents of the invention can be determined empirically and can beemployed in pure form or, where such forms exist, in pharmaceuticallyacceptable salt, ester or prodrug form. The agents can be administeredto a patient in order to prevent and/or reduce ototoxicity resultingfrom ototoxic agents, noise or head and neck radiation as pharmaceuticalcompositions in combination with one or more pharmaceutically acceptableexcipients. It will be understood that, when administered to a humanpatient, the total daily usage of the agents or composition of thepresent invention will be decided by the attending physician within thescope of sound medical judgment. The specific therapeutically effectivedose level for any particular patient will depend upon a variety offactors: the type and degree of the cellular or physiological responseto be achieved; activity of the specific agent or composition employed;the specific agents or composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the agent; theduration of the treatment; drugs used in combination or coincidentalwith the specific agent; and like factors well known in the medicalarts. For example, it is well within the skill of the art to start dosesof the agents at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosages until thedesired effect is achieved.

One skilled in the art can also readily determine an appropriate dosageregimen for administering the siRNA of the invention to a given subject.For example, the siRNA can be administered to the subject once, such asby a single injection or deposition. Alternatively, the siRNA can beadministered to a subject multiple times daily or weekly. For example,the siRNA can be administered to a subject once weekly for a period offrom about three to about twenty-eight weeks, more preferably from aboutseven to about ten weeks. The administration regimen will also depend onthe cause of ototoxicity and exposure of the patient to any of theototoxic agents, noise or head and neck radiation. The siRNAs of thepresent invention can either be administered prophylactically, e.g.,before starting the aminoglycoside regimen, during exposure of thepatient to the ototoxic environment, following such exposure, or acombination thereof. By way of example, a siRNA directed against TRPV1or NOX3 can be administered prior to the start of cisplatin treatment,during such treatment, and optionally following such treatment. By wayof another example, a siRNA against TRPV1 or NOX3 can be administeredprior to the start of an aminoglycoside treatment, during suchtreatment, and optionally following such treatment. When administeredafter the drug treatment or noise exposure, it is preferable that thesiRNA of the present invention be administered within a reasonable timethereafter, e.g., such as a few weeks.

In another embodiment, the siRNA of the present invention can beadministered in combination with one or more agents used to treatototoxicity, such as sodium thiosulfate, D- or L-methionine,diethyldithiocarbamate, methylthiobenzoic acid, lipoic acid,N-acetylcysteine, thiopronine, glutathione ester, and amifostine. Thus,by way of example, a siRNA directed against TRPV1 or NOX3 can beadministered in combination with D-methionine.

In another embodiment, the siRNA of the present invention can be used toprevent or reduce generation of reactive oxygen species in the inner earof a patient. As described above, at least one siRNA selected fromsiRNAs directed against TRPV1 and NOX3 can be used. Furthermore, sincethe generation of reactive oxygen species plays a role in tinnitus, thepresent invention provides a method for treating a patient sufferingfrom tinnitus and a method for preventing and/or reducing ototoxicity ina patient suffering from or at risk for developing ototoxicityassociated with tinnitus. For methods of treating tinnitus, theselection of siRNAs, their administration and dosage is the same or canbe determined as described above.

Treatment of Nephrotoxicity

Nephrotoxicity can be induced by aminoglycoside antibiotics and byplatinum-containing drugs such as cisplatin. As can be expected,nephrotoxicity has important consequences for the patient, withpotential permanent loss of 50% or more of normal renal function (Kemp,et al. J. Clin. Oncology, 14:2101-2112, 1996). This can produce seriousdisability, requiring the need for dialysis in severe cases, and earlymortality. It also has important consequences for the ability of thepatient to be safely treated with medications such as antibiotics thatare themselves renally toxic or require adequate renal function forelimination from the body. Thus, it is contemplated that siRNAs againstTRPV1 and NOX3 can also be used to treat nephrotoxicity in a patient,wherein the nephrotoxicity is caused by administration ofaminoglycosides or platinum-containing chemotherapeutic agents. In thisembodiment, at least one siRNA of the present invention can beadministered by any method known in the art that will efficiently resultin the presence of the siRNA(s) in the kidney. By way of example, atleast one siRNA of the present invention can be administeredintravenously or intraperitoneally. In one preferred embodiment, atleast one siRNA directed against TRPV1 is administered, e.g.,intravenously. In another preferred embodiment, at least one siRNAdirected against NOX3 is administered, e.g., intravenously. In stillanother preferred embodiment, a combination of at least one siRNAdirected against TRPV1 and at least one siRNA directed against NOX3 isadministered, e.g., intravenously.

General Methods

Molecular biological techniques, biochemical techniques, andmicroorganism techniques as used herein are well known in the art andcommonly used, and are described in, for example, Sambrook J. et al.(1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor andits 3rd Ed. (2001); Ausubel, F. M. (1987), Current Protocols inMolecular Biology, Greene Pub. Associates and Wiley-interscience;Ausubel, F. M. (1989), Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-interscience; Innis, M. A. (1990), PCRProtocols: A Guide to Methods and Applications, Academic Press; Ausubel,F. M. (1992), Short Protocols in Molecular Biology: A Compendium ofMethods from Current Protocols in Molecular Biology, Greene Pub.Associates; Ausubel, F. M. (1995), Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates; Innis, M. A. et al. (1995), PCR Strategies,Academic Press; Ausubel, F. M. (1999), Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, and annual updates; Sninsky, J. J. et al. (1999), PCRApplications: Protocols for Functional Genomics, Academic Press; Specialissue, Jikken Igaku [Experimental Medicine] “Idenshi Donyu &Hatsugenkaiseki Jikkenho [Experimental Method for Gene introduction &Expression Analysis]”, Yodo-sha, 1997; and the like. Relevant portions(or possibly the entirety) of each of these publications are hereinincorporated by reference.

Any technique may be used herein for introduction of a nucleic acidmolecule into cells, including, for example, transformation,transduction, transfection, and the like. Such a nucleic acid moleculeintroduction technique is well known in the art and commonly used, andis described in, for example, Ausubel F. A. et al., editors, (1988),Current Protocols in Molecular Biology, Wiley, New York, N.Y.; SambrookJ. et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. and its3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Special issue, Jikken Igaku [Experimental Medicine] Experimental Methodfor Gene introduction & Expression Analysis”, Yodo-sha, 1997; and thelike. Gene introduction can be confirmed by method as described herein,such as Northern blotting analysis and Western blotting analysis, orother well-known, common techniques.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing the scope ofthe invention defined in the appended claims. Furthermore, it should beappreciated that all examples in the present disclosure, whileillustrating the invention, are provided as non-limiting examples andare, therefore, not to be taken as limiting the various aspects of theinvention so illustrated.

Examples

The following non-limiting examples are provided to further illustratethe present invention. It should be appreciated by those of skill in theart that the techniques disclosed in the examples that follow representapproaches the inventors have found function well in the practice of theinvention, and thus can be considered to constitute examples of modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Methods

Animal procedures and sample collection: Male Wistar rats were used forthis study. Pre-ABR's were performed 2-3 days before round windowapplication of siTRPV1 or PBS. 48 hrs post surgery, cisplatin (13 mg/kg)was administered by intraperitoneal injections over a period of 30 minand sacrificed at 24, 48 or 72 h following treatment. The cochleas weredissected and used for the preparation of total RNA or total proteinextracts, or perfused with 2.5% glutaraldehyde for morphological studiesby Scanning Electron Microscopy (SEM's) or 4% paraformaldehyde forimmunocytochemistry.

Measuring Evoked Potentials

Auditory brainstem responses were measured pre-surgery and 72 his postcisplatin treatment as described in (Tanaka, et al. 2003, pH paper).Animals were tested with a stimulus intensity series that was initiatedat 10 dB SPL and reached a maximum at 90 dB SPL. The stimulus intensitylevels were increased in 10 dB increments, and the evoked ABR waveformswere observed on a video monitor. The auditory stimuli included tonebursts at 2, 4, 8, 16 and 32 kHz with a 10 msec plateau and a 1 msecrise/fall time presented at a rate of 5 per second. Threshold wasdefined as the lowest intensity capable of evoking a reproducible,visually detectable response with two distinct waveforms and a minimumamplitude of 0.5 μV. The pretreatment ABR thresholds were compared topost-treatment thresholds and the differences were evaluated forstatistical significance using the Student's t-test.

Morphological Studies: Scanning Electron Microscopy

Immediately after completion of follow-up ABRs, deeply sedated rats wereeuthanized, their cochleae harvested and processed as described in(Kamimura et al, 1999). Sputter coated cochleae were then viewed andphotographed with a Hitachi S-500 scanning electron microscope (HitachiLtd., Tokyo, Japan).

Processing of Cochlea for Immunocytochemistry

Cochleae perfused with 4% paraformaldehyde were processed fordecalcification and sectioning as described in (Dunaway et al., 2003).TRPV1-1 antibody was diluted 1:100 and samples were incubated for 1 h at37° C. incubator. Secondary antibody used was goat anti-rabbit IgGconjugated to horse radish peroxidase (Santa Cruz Biotechnology, Inc,Santa Cruz, Calif.) which was diluted 1:200. ABC staining system (SantaCruz Biotechnology), which included a diaminobenzadine as a peroxidasesubstrate, was used for visualization of protein expression. Slides wereimaged using Scion Imaging system (Frederick, Md.).

Hair Cell Count

Hair cell counts were performed using a modified version of the methodused by (Korver et al. 2002). Two representative areas of the basal turnand hook portion were photographed. In each area, inner or outer haircells were counted in an area that was 10 pillar cell heads in length.

The results were shown, as the average survival percentage ratescompared to the control group. Statistical analysis was performed usingStudent's t-test.

Cell Cultures

Immortalized organ of Corti cells derived from the mouse, UB/OC-1 cells,were obtained from Dr. Matthew Holley (Institute of MolecularPhysiology, Addison Building, Western Bank, Sheffield, UK) and culturedin RPMI 1640 supplemented with 10% Fetalclone II (Hyclone, Logan, Utah)serum and penicillin-streptomycin. Cultures were grown in a 33° C.incubator in 10% CO2.

Reagents

The various reagents: cisplatin, diphenyleneiodonium (DPI), AEBSFCapsezapine, Capsaicin, Ruthinium red, TR1 reagent and,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) werepurchased from (Sigma-aldrich, St. Louis, Mo.). Frag-EL DNAfragmentation kit and H2DCFDA dye was purchased from (Calbiochem, SanDiego, Calif.). TRPV1 antibody (Neuromics, Edina, Minn.) and secondarygoat anti rabbit antibody was purchased from Santa Cruz Biotechnology(Santa Cruz, Calif.).

RNA Isolation

RNA was isolated by adding 1 ml TR1 reagent to 100 mg of cochlear orcortical tissue or 0.5 ml TR1 reagent (Sigma, St. Louis, Mo.) per wellof each six well plate, according to the manufacturer's instructions.

Real time reverse transcriptase polymerase chain reaction (RT-PCR): Onemicrogram of total RNA was converted to cDNA using iScript cDNASynthesis Kit (Bio-Rad, Hercules, Calif.), and qRTPCR was performed asdescribed by (Mukherjea et al, 2006). Gene specific primer pairs wereused for the various reactions and mRNA expression levels werenormalized to the levels of GAPDH house keeping gene.

Oligonucleotides

The rodent set of primers and siRNA were based on the homologoussequences in the rat and mouse cDNA sequences. The primers werepurchased from Sigma Genosys (St. Louis, Mo.). Purified siRNA duplexeswere purchased from Qiagen (Valencia, Calif.). Primers were as follows:Rodent NOX3 (sense): 5′-GTG AAC AAG GGA AGG CTC AT-3′ (SEQ ID NO: 9)(antisense): 5′-GAC CCA CAG AAG AAC ACG C-3′ (SEQ ID NO: 10),Rodent-GAPDH (sense): 5′-ATG GTG AAG GTC GGT GTG AAC-3′ (SEQ ID NO: 11)(antisense): 5′-TGT AGT TGA GGT CAA TGA AGG-3′ (SEQ ID NO: 12), RodentTRPV1 (sense): 5′-CAA GGC TGT CTT CAT CAT CC-3′ (SEQ ID NO: 13),(antisense): 5-AGT CCA GTT TAC CTC GTC CA-3′ (SEQ ID NO: 14), RodentRac-1 (sense): 5′-ATC AGT TAC ACG ACC AAT GC-3′ (SEQ ID NO: 15),(antisense): 5′-GGG AAA AGC AAA TTA AGA AC-3′ (SEQ ID NO: 16), Rodentgp-91 (sense) 5′-TAA AGG AGT GCC CAG TAC CAA-3′ (SEQ ID NO: 17),(antisense): 5′-AAT CCC TTC TTC TTC ATC TGA-3′ (SEQ ID NO: 18), and Ratp22 (sense): 5′-ACA GGG GGC ATC GTG GCT ACT-3′ (SEQ ID NO: 19),(antisense) 5′-GGA CGT AGT AAT TTC TGG TGA-3′ (SEQ ID NO: 20)

Rodent siNOX3: Target sequence: 5′-AAGGTGGTGAGTCACCCATCT-3′ (SEQ ID NO:8)

Rodent siTRPV1: Target sequence: 5′-GCGCATCTTCTACTTCAACTT-3′ (SEQ ID NO:7)

A sense strand of siRNA from Christoph et al. (2006)5′GCGCAUCUUCUACUUCAACTT-3′ (SEQ ID NO: 2)

A sense strand of siRNA against TRPV1: GGU GGU GAG UCA CCC AUC UdTdT(SEQ ID NO: 1)

A sense strand of siRNA against NOX3: GGU GGU GAG UCA CCC AUC U)dTdT(SEQ ID NO: 3)

Western Blot Analysis

Cochleae were homogenized in ice-cold TRIS 50:10:100. The whole tissuelysate was then used for Western blotting. After transfer tonitrocellulose membrane TRPV1 protein was visualized by chemiluminesencedetection (ECL, Amersham, Piscataway, N.J.).

Results

Cisplatin

For these studies, male Wistar rats (200-250 g) were administeredvehicle or cisplatin (13 mg/kg) by intraperitoneal infusion over a 30min period. Rats were tested for hearing loss 72 h later using auditorybrainstem evoked responses (ABRs). Cisplatin produced a significantelevation in auditory evoked brain stem responses by 20-40 dB over awide frequency range (8-32 kHz) (as described in Whitworth et al.,Biochem Pharmacol., 67, 1801-1807, 2004; Mukherjea et al., Neuroscience,139, 733-740, 2006). Cochleas obtained from these animals weredecalcified, sectioned and processed for TRPV1 immunoreactivity using apolyclonal antibody (as described in Puntambekar et al., 2005).Visualization by confocal microscopy indicated TRPV1 immunoreactivity inthe organ of Corti, supporting cells and spiral ganglion cells. Bothinner and outer hair cells showed immunolabeling. TRPV1immunoreactivity, as determined by confocal microscopy, was elevated inhair cells and spiral ganglion cells by 2-3 fold by 72 h followingcisplatin treatment (FIG. 1A). No labeling was obtained if the antibodywas not added to the immunolabeling mix, suggesting antibody-specificlabeling. The increase in immunolabeling observed at 72 h wassubstantiated by Western blotting studies showing increased TRPV1protein (˜95 kDa band) in whole cochlear lysates (FIG. 1B). Theincreases in TRPV1 immunoreactivity were associated with a 15±1-foldincrease in TRPV1 transcripts by 24 h following cisplatin treatment,with no further elevations by 48 and 72 h. The increases were 18±1 and15±3 at 48 h and 72 h, respectively (FIG. 1C). These increases in TRPV1transcripts preceded morphological changes in hair cell, which aregenerally observed by 72 h following cisplatin administration (as shownby Ford et al., Hear Res., 111, 143-152, 1997 and Whitworth et al.,2004, supra) and which involve ROS generation (Kopke, Am. J. Otol., 18,559-571, 1997). To determine whether ROS generation could be implicatedin TRPV1 induction, as demonstrated previously (Puntambekar et al., J.Neurochem., 1689-1703, 2005), the effect of the antioxidant, lipoicacid, was tested on cisplatin-induced TRPV1 expression in vivo. Eventhough the level of induction of TRPV1 by cisplatin (5.5±1.5-fold) wassmaller than obtained previously, it was completely abolished in ratspretreated with lipoic acid (FIG. 1D). Lipoic acid also showed atendency to reduce basal expression of TRPV1 (albeit not statisticallysignificant), implicating ROS in this response.

The NOX3 isoform of NADPH oxidase represents the most abundant form ofthis enzyme present in the cochlea, whose expression is induced bycisplatin in organotypic cultures (Banfi et al., J. Biol. Chem., 279,46065-46072, 2004). Results shown in FIG. 1E indicate a small butstatistically significant increase in NOX3 transcript in the cochlea bycisplatin. This induction was close to maximum by 24 h, with no furthersignificant change observed by 48 h and a reduction by 72 h (FIG. 1F).The increase in NOX3 by cisplatin was attenuated by lipoic acid,implicating ROS in its induction (FIG. 1G). In addition, lipoic acidsignificantly reduced the basal expression of NOX3, implicating ROS inthis process. Other NADPH oxidase isoforms, such as Rac1, gp91 and p22,were also induced by cisplatin, with increases in expression being478±85, 1000±102 and 8±1 fold, respectively (data not shown). The highfold induction of these latter transcripts over that observed for NOX3might reflect their low basal expression in the cochlea as compared toNOX3.

To further study the role of ROS generated via cochlear NADPH oxidasesin the induction of TRPV1 in the cochlea, in vitro experiments wereperformed using the organ of Corti transformed hair cell line, UB/OC1(Rivolta et al., Proc. R. Sco. Lond., 265, 1595-1603, 1998). These cellshave been used previously to examine cisplatin induction of the kidneyinjury molecule (KIM-1) protein (Mukherjea et al., 2006, supra).Treatment of UB-OC1 cells with cisplatin (2.5 μM) for 24 h resulted in a71±29% induction in TRPV1 immunoreactivity over control vehicle treatedcells (FIG. 2 b). Immunolabeling was quantitated by fluorescence imagingusing a confocal microscope. Pretreatment of these cultures with either100 μM AEBSF (Diatchuk et al., J. Biol. Chem., 272(20), 13292-13301,1997) or 10 μM DPI (O'Donnell et al., Biochem J., 290, 41-49, 1993),inhibitors of NADPH oxidase, attenuated the increase observed withcisplatin, implicating ROS in this process (FIG. 2A). At theseconcentrations, these inhibitors did not significantly affect cellviability over a 24 h period. The immunofluorescence in these groups wasreduced by 60 and 70%, respectively (FIG. 2B). The increase in TRPV1protein expression produced by cisplatin in UB-OC1 cells was confirmedby Western blotting which showed a significant increase in TRPV1 protein(FIG. 2C) by 90%±10% (FIG. 2D). In addition, a significant increase inTRPV1 mRNA was observed in 24 h (FIG. 2E). UB-OC1 cells treated withcisplatin showed a robust increase in ROS generation, as determined byDCF2DA fluorescence. This increase was abolished in cells pretreatedwith AEBSF (100 μM), indicating of a role of NADPH oxidase activation(and possibly of NOX3) in the ROS generation induced by cisplatin (FIG.2F). The increase in ROS generation was followed by a significantincrease in mRNA encoding different NADPH oxidase subunits. Astatistically significant increase in NOX3, gp91 and Rac1 by 2.7, 2.8and 2.1 fold, respectively, was observed. The increases in expression ofthese genes were inhibited by DPI (10 μM), indicative of a role or NADPHoxidase activity in the induction of these subunits (FIG. 2G).

Treatment of UB-OC1 cells with a higher concentration of cisplatin (20μM) for 24 h promoted apoptosis, as detected by TUNEL staining. At thisconcentration of cisplatin, ˜80% of apoptotic cells were obtained inUB-OC1 cultures. UB-OC1 cells pretreated for 30 min with eithercapsazepine or ruthenium red, inhibitors of TRPV1, and then administeredcisplatin, showed significantly less apoptotic cells (˜7% of totalcells) (FIGS. 2H,I). Neither capsazepine nor ruthenium red alone had anysignificant effect on cell apoptosis, compared to vehicle-treatedcontrol groups. These findings suggest that TRPV1 is an importantintermediary for mediating cisplatin apoptosis in UB-OC1 cells. Theincrease in apoptosis by cisplatin was associated with increases inproapoptotic proteins such as p53 and Bax (FIGS. 2J,K). The increases inthese proteins were attenuated by capsazepine (10 μM), implicating TRPV1in this process. Cells pretreated with BAPTA-AM for 30 min prior theadministration of cisplatin showed a substantial reduction in Baxprotein, suggesting a role of intracellular Ca2+ in mediating apoptosisinduced by cisplatin. Using Fura4-AM, it was shown that cisplatinincreased intracellular Ca2+ in UB/OC-1 cells, which was inhibited usingsiRNA against TRPV1. This observation suggests that activation of TRPV1by cisplatin results in intracellular Ca2+ accumulation, which cantrigger apoptosis of UB/OC-1 cells.

It was next determined whether selective inhibition of NOX3 synthesis byshort interfering (si) RNA would reduce the expression of TRPV1 andpossibly NOX3 itself. It was observed that NOX3 siRNA reduced the basaland cisplatin-stimulated expression of NOX3 by 24 h, without grosslyaffecting the morphology of the cells. Quantitation of TRPV1 mRNA byreal time PCR indicated that NOX3 siRNA significantly reduced the basaland cisplatin-induced TRPV1 expression (FIG. 3A). Based on the previousdata using inhibitors of NADPH oxidase (in vitro) and lipoic acid (invivo), these data implicated NOX3 as a regulator of TRPV1 in UB-OC1cultures. Interestingly, when cells were incubated with siRNA againstTRPV1, attenuation of both the cisplatin-induced TRPV1 and NOX3expression was observed, indicating possible co-regulation of theseproteins (FIG. 3A).

To determine whether administration of TRPV1 siRNA would be effectiveagainst cisplatin-induced hearing loss, this agent was administered byround window application for 48 h and subsequently administeredintraperitoneal cisplatin (13 mg/kg). Animals were tested 72 h later forauditory functions. Using cyanine-3 labeled siRNA, it was shown thatround window application resulted in delivery of the fluorescent siRNAinto the organ of Corti by 3 days after administration (the earliesttime examined) and the signal persisted for ˜10 days. A significantincrease in ABR thresholds was observed following cisplatin treatmentalone which averaged 20-40 dB over an 8-32 kHz frequency range. However,in rats pre-treated with TRPV1 siRNA (0.9 μg/3 μl) 48 hrs prior tocisplatin administration, there was no significant shift in ABRthreshold, implying effective protection against cisplatin ototoxicity(FIG. 3B). Significant protection against hearing loss was observed at 8and 16 kHz tones, while a trend towards protection was observed using a32 kHz tone (FIG. 3B). Real time PCR performed to determine the level ofreduction in TRPV1 mRNA in the cochlea indicated an ˜85% decrease inexpression following administration of a single concentration of TRPV1siRNA and examining the cochlea on day 3 (FIG. 3C). In addition, asignificant reduction of TRPV1 protein levels was observed in cochleasharvested from rats administered TRPV1 siRNA over the same time period(FIGS. 3D,E).

Morphological assessment of the outer hair cells by scanning electronmicroscopy indicated that cisplatin produced significant damage or lossof hair cells. Five rats were administered either vehicle or TRPV1 siRNA(3 μl) by round window application. The rats served as their owncontrols since one cochlea was treated with vehicle and the other withsiRNA. Twenty four hours following the application of vehicle or siRNA,rats were infused with cisplatin (16 mg/kg, i.p) over a 30 min periodand assessed for hair cell damage 3 days later. In 3 of the 5 ratstreated with cisplatin, significant loss or damage of outer hair cellswas observed in the cochlea pretreated with vehicle prior to cisplatin,but significant protection in the TRPV1 siRNA treated cochleas (FIG.3F). The percentage of outer hair cell loss in the hook, base and middleturn of the cochlea in rats administered cisplatin (16 mg/kg, i.p.) was72±14, 42±9 and 22±2.7 respectively (N=3). However, pretreatment withTRPV1 siRNA resulted in a significant reduction in percentage of haircell loss to 21±12, 14±21 and no loss in the hook, base and the middleturns respectively (N=3) (statistically significant difference, p≦50.05)(FIG. 3G). One animal showed partial protection on the siRNA pretreatedside, while no protection was observed in the cochlea of the remaininganimal. No significant change in outer hair cell morphology was observedfollowing the administration of TRPV1 siRNA alone (FIG. 3G).

Noise

Prior to use, the animals were anesthetized and their heads wereimmobilized in a small animal stereotaxic with hollow earbars (DavidKopf Instruments, Tujunga, Calif.). Animals were placed in adouble-walled sound attenuating radio-frequency shielded booth(Industrial Acoustics Corporation) and their body temperatures weremaintained at 37° C. using an animal warming blanket. Subdermal platinumalloy needle electrodes were attached with the active lead at the vertexand referred to a second electrode located over the temporal bone. Theground electrodes were placed over the neck muscles. Ten millisecondtone bursts (1, 2, 4, 8 and 16 kHz) were delivered monaurally throughetymotic insert earphone placed directly into the ear bar. Auditorystimuli were presented at a rate of five per second with increasingintensity from 30 to 90 decibels (dB) sound pressure level (SPL) in 10dB steps. The responses were amplified 100,000 times before beingrecorded. Auditory brainstem response (ABR) waveforms were repeated foreach intensity. The response that clearly showed a reproducible waveform that displayed two or more peaks with a minimum amplitude of 0.5 μVwas interpreted as the threshold response.

Noise exposure (90 dB, 6 h) produced a threshold shift in the ABR(between 8 and 16 kHz), which was observed when animals were testedafter noise exposure. The shifts ranged between 38-42 dB at the twofrequencies tested (FIG. 4A).

In order to determine whether noise exposure increased the activity ofNADPH oxidase in the chinchilla cochlea, a major source of reactiveoxygen species (ROS), the activity of NADPH oxidase in cytosolicpreparations from the cochlea was examined. The activity of NAD(P)Hoxidase was determined using 50 μg protein from cochlear extractsincubated with 100 μM β-NAD(P)H in a total volume of 200 μl of Hank'sHEPES buffer (pH 7.4). The assay was initiated by the addition of 50 μMN,N′-dimethyl-9,9′-biacridinium dinitrate (Lucigenin) (Sigma, St. Louis,Mo.) to the incubation mixture. Samples were counted immediately using atable top luminometer (Berthold Detection Systems FB Luminometer, ZyluxCorp., Maryville, Tenn.) with sampling time every 6 sec. Samples werecounted over a period of 5 min and the fluorescence recorded over 2 minof stable readings were averaged for that samples. Samples were run induplicate and the NAD(P)H oxidase activity was normalized to the proteinconcentrations in each sample.

Initial characterization of NADPH oxidase activity usingN,N′-dimethyl-9,9′-biacridinium dinitrate (Lucigenin) as the fluorescentprobe indicate blockade of enzyme activity by 10 μM diphenyleneiodoniumchloride (Sigma, St. Louis, Mo.), a known inhibitor of this enzyme.Enzyme activity was greater using NADPH (100 μM) as a co-factor thanwith NADH (100 μM), indicative of NADPH oxidase (FIG. 4B).

Cochlear homogenates prepared from noise exposed animals (chinchillawere exposed to 96 dB noise for 6 h) demonstrated higher levels of NADPHoxidase activity, which were evident soon after the first noise exposureperiod (days 1) and on day 3 (i.e. 3 consecutive daily noise exposures).Cochlear NADPH oxidase activity increased from 278±27 (control ear) to457±58 arbitrary units, following noise exposure for 1 day. Separateexperiments were performed using different controls and 3 day noiseexposed cochleae. These samples indicated that the NADPH oxidaseactivity increased from 338±23 (control ear) to 520±117 arbitrary units(FIG. 4C).

Additional experiments were performed in rats to determine whether theincrease in NADPH oxidase activity observed with noise was associatedwith increased expression of NADPH oxidase subunits and TRPV1. Usingprimers for Rac1, NOX3 and TRPV1 in real time PCR assays, we observedstatistically significant increases (˜8-10 fold) in the Rac1 and NOX3subunits of NADPH oxidase and in TRPV1 (FIG. 4D).

Aminoglycosides

In this study, the mouse organ of Corti cell line (UB/OC-1) was used toexamine the properties of aminoglycoside-induced ROS generation. Forthese experiments, cells were grown on coverslips to 60-80% confluency,then pretreated with vehicle (control and gentamicin groups),capsazepine (10 μM) or ruthenium red (20 μM) for 15 min. This wasfollowed by vehicle (control) or gentamicin for 30 min. Cells were thenloaded with 2′,7′-dichlorodihydrofluorescein diacetate (DCF2DA) andimaged by confocal microscopy. As shown in FIG. 5, the ROS generation ofthe gentamicin group was suppressed by capsazepine and ruthenium red(FIG. 5).

1. A method for preventing and/or reducing ototoxicity in a patientsuffering from or at risk for developing ototoxicity caused by anototoxic agent, noise, head or neck radiation, or tinnitus wherein themethod comprises silencing TRPV1 in the patient.
 2. The method of claim1, wherein the silencing of the TRPV1 mRNA is achieved by administeringsiRNA directed against TRPV1 mRNA.
 3. The method of claim 2, wherein asense strand of the siRNA directed against TRPV1 mRNA comprises anucleic acid sequence of SEQ ID NO:
 1. 4. The method of claim 2, whereina sense strand of the siRNA directed against TRPV1 mRNA comprises asense nucleic acid sequence of SEQ ID NO:
 2. 5. The method of claim 1,wherein the ototoxic agent is selected from the group consisting of anaminoglycoside and a platinum-containing chemotherapeutic agent.
 6. Themethod of claim 5, wherein the aminoglycoside is selected from the groupconsisting of neomycin, paromomycin, ribostamycin, lividomycin,kanamycin, amikacin, tobramycin, viomycin, gentamicin, sisomicin,netilmicin, streptomycin, dibekacin, fortimicin, dihydrostreptomycin,and a combination thereof.
 7. The method of claim 5, wherein theplatinum-containing chemotherapeutic agent is selected from the groupconsisting of cisplatin and carboplatin.
 8. The method of claim 7,wherein the platinum-containing chemotherapeutic agent is cisplatin. 9.The method of claim 2, wherein the siRNA directed against TRPV1 mRNA isadministered locally.
 10. The method of claim 9, wherein the siRNAdirected against TRPV1 mRNA is administered to round window orintra-tympanically.
 11. A method for preventing and/or reducingototoxicity in a patient suffering from or at risk for developingototoxicity caused by an ototoxic agent, noise, head or neck radiation,or tinnitus wherein the method comprises administering to the patient atleast one siRNA selected from siRNAs directed against TRPV1 mRNA andNOX3 mRNA.
 12. The method of claim 11, wherein the at least one siRNAdirected against TRPV1 mRNA and at least one siRNA directed against NOX3mRNA are administered to the patient.
 13. The method of claim 11,wherein a sense strand of the siRNA directed against TRPV1 comprises anucleic acid sequence selected from SEQ ID NO: 1 or SEQ ID NO:
 2. 14.The method of claim 11, wherein a sense strand of the siRNA directedagainst NOX3 mRNA comprises SEQ ID NO:
 3. 15. The method of claim 11,wherein the ototoxic agent is selected from the group consisting of anaminoglycoside and a platinum-containing chemotherapeutic agent.
 16. Themethod of claim 15, wherein the aminoglycoside is selected from thegroup consisting of neomycin, paromomycin, ribostamycin, lividomycin,kanamycin, amikacin, tobramycin, viomycin, gentamicin, sisomicin,netilmicin, streptomycin, dibekacin, fortimicin, dihydrostreptomycin,and a combination thereof.
 17. The method of claim 15, wherein theplatinum-containing chemotherapeutic agent is selected from the groupconsisting of cisplatin and carboplatin.
 18. The method of claim 17,wherein the platinum-containing chemotherapeutic agent is cisplatin. 19.The method of claim 11, wherein the at least one siRNA is administeredlocally.
 20. The method of claim 19, wherein the at least one siRNA isadministered to round window or intra-tympanically.
 21. A method forpreventing or reducing generation of reactive oxygen species in an innerear of a patient, the method comprising administering to the patient atleast one siRNA selected from the group consisting of a siRNA directedagainst TRPV1 mRNA and siRNA directed against NOX3 mRNA.
 22. The methodof claim 21, wherein a sense strand of the siRNA directed against TRPV1mRNA comprises a nucleic acid sequence of SEQ ID NO:
 1. 23. The methodof claim 21, wherein a sense strand of the siRNA directed against TRPV1mRNA comprises a nucleic acid sequence of SEQ ID NO:
 2. 24. The methodof claim 21, wherein a sense strand of the siRNA directed against NOX3mRNA comprises a nucleic acid sequence of SEQ ID NO:
 3. 25. The methodof claim 21, wherein the generation of reactive oxygen species resultsfrom treatment of the patient with an ototoxic agent selected from thegroup consisting of an aminoglycoside and a platinum-containingchemotherapeutic agent.
 26. The method of claim 25, wherein theaminoglycoside is selected from the group consisting of neomycin,paromomycin, ribostamycin, lividomycin, kanamycin, amikacin, tobramycin,viomycin, gentamicin, sisomicin, netilmicin, streptomycin, dibekacin,fortimicin, dihydrostreptomycin, and a combination thereof.
 27. Themethod of claim 25, wherein the platinum-containing chemotherapeuticagent is selected from the group consisting of cisplatin andcarboplatin.
 28. The method of claim 27, wherein the platinum-containingchemotherapeutic agent is cisplatin.
 29. The method of claim 21, whereinthe at least one siRNA selected from siRNA directed against TRPV1 mRNAand NOX3 mRNA is administered locally.
 30. The method of claim 29,wherein the at least one siRNA selected from siRNA directed againstTRPV1 mRNA and NOX3 mRNA is administered to round window orintra-tympanically.